1. Field of the Invention
This invention relates to a novel reagent composition and its use in an assay for the determination of .gamma.-glutamyltransferase activity in serum and other biological samples.
2. Nature and Usefulness of .gamma.-Glutamyltransferase Determinations
Determinations of the quantity of the enzyme .gamma.-glutamyltransferase, in human serum, are being performed in the clinical laboratory with ever increasing frequency. Such determinations appear to be the most sensitive screening test for liver disease currently available, since, almost without exception, a normal level of .gamma.-glutamyltransferase denotes a healthy liver. The measurement of .gamma.-glutamyltransferase is extremely useful to the physician in determining the extent of liver damage caused by chronic alcoholism, and in monitoring the progress of viral hepatitis. Levels of .gamma.-glutamyltransferase are also increased in acute pancreatitis, in certain cardiovascular diseases, and in kidney failure; therefore, although the enzyme .gamma.-glutamyltransferase has been known only since 1950, and it was not until the late 1960's that the development of new methodology for .gamma.-glutamyltransferase determination permitted its full significance as a valuable diagnostic tool to become recognized and accepted, its determination is a most valuable part of any clinical testing profile. Correspondingly, the ease and other factors of the assays for this significant enzyme have been increasingly a matter of concern to the large and active field of diagnostic bio-medical assays.
3. Description of the Prior Art
While the prior art has specified several methods in its search of assays to determine .gamma.-glutamyltransferase activity, the methods generally considered suitable for use in the clinical laboratory have been those which use a synthetic .gamma.-glutamyl substrate with chromogenic properties; for it is known that the enzyme .gamma.-glutamyltransferase catalyzes the transpeptidation or transfer of .gamma.-glutamyl groups from .gamma.-glutamyl peptide substrates to suitable acceptors.
However, some of these substrates require the analyst to perform cumbersome and time-consuming procedural steps, and some of these substrates are considered to be carcinogenic substances and are therefore extremely dangerous for use by laboratory personnel. The method which utilizes .gamma.-glutamyl-p-nitroanilide as the substrate and glycylglycine as the acceptor is therefore generally considered to be the safest and most practical of the various .gamma.-glutamyltransferase assay methods, and may be described by the following reaction sequence: ##STR1##
Unfortunately, however, there are two serious disadvantages hindering the routine and frequent use of this otherwise acceptable method, namely, the instability and poor solubility of the .gamma.-glutamyl-p-nitroanilide substrate. It is not only difficult to dissolve the compound, but once solvation has been accomplished, the resulting solution remains usable for only a short period of time, usually no longer than two or three hours due to decomposition of the .gamma.-glutamyl-p-nitroanilide. This problem is further exacerbated by the fact that any attempt to prolong stability by refrigeration of the solution will cause the substrate to crystallize and precipitate out of solution. It has generally been necessary to use temperatures as high as 50.degree. to 60.degree. C. in order to dissolve .gamma.-glutamyl-p-nitroanilide; however, such temperatures are very close to that at which .gamma.-glutamyl-p-nitroanilide will spontaneously hydrolyze, which obviously has a deleterious effect on the usefulness of the solution.
Carroll, in U.S. Pat. Nos. 3,769,173 (Oct. 30, 1973), 3,878,048 (Apr. 15, 1975), and 3,892,631 (July 1, 1975) describes a substrate composition wherein, in addition to the .gamma.-glutamyl-p-nitroanilide and glycylglycine, there is also included sodium nitrite, an ammedial buffer, and, optionally, magnesium chloride hexahydrate. Carroll suggests that the magnesium chloride may help to keep the substrate in solution, but this was the extent of his teaching with regard to stabilizing .gamma.-glutamyl-p-nitroanilide. Carroll's assay method, however, required three working reagents and several procedural steps, and thus would be considered too cumbersome and time-consuming to meet the needs of today's clinical laboratory and its need for high-volume and automated testing capabilities.
Bernt et al. in U.S. Pat. No. 3,979,447 (Sept. 7, 1976) and in U.S. Pat. No. 3,986,931 (Oct. 19, 1976) describe certain .gamma.-glutamyl-p-nitroanilide compounds characterized by an acid group substituted at position 3 of the nitroaniline, which are claimed to help overcome the problems of instability and poor solubility. The preferred substrates of the Bernt et al. art are specified to be .gamma.-glutamyl-3-carboxy-p-nitroanilide and .gamma.-glutamyl-3-sulfo-p-nitroanilide. Indeed, these compounds do exhibit improved solubility characteristics and tend to precipitate out of solution less easily than the unsubstituted compound, and in this respect, stability is therefore improved.
However, the Bernt et al. art, other than asserting that the "stability is good", does not even venture to suggest any particular duration of stability; and it seems justified to presume that there still remained some drawbacks, at least with regard to stability, since in their teachings as to how to perform an assay, Bernt et al. chose to keep the substrate solution separate from the buffer solution and use two reagent pipetting steps to perform an assay. Furthermore, the preferred substrates of Bernt et al. have been found to exhibit diminished sensitivity when compared to that seen with unsubstituted .gamma.-glutamyl-p-nitroanilide.
Nakanishi et al. in U.S. Pat. No. 3,703,441 (Nov. 21, 1972) attempted to improve the solubility and stability of .gamma.-glutamyl-p-nitroanilide by including a combination of a particular surface active agent and a particular buffer. In their teachings, even a solution as strong as 30 percent dimethylsulfoxide was dismissed as being unsuitable as a solvent. Quite unexpectedly, therefore, it was found that as little as 1 percent dimethylsulfoxide was significantly effective in enhancing the stability of a solution containing .gamma.-glutamyl-p-nitroanilide and .beta.-cyclodextrin, especially at refrigerated storage temperatures.